In this project we focus our studies on the genetic basis both of orthopoxvirus virulence and of host resistance to virus infection. The acquired knowledge should contribute towards the development of safe, effective, recombinant vaccinia virus vaccines for animal and human use. In this year, two separate approaches have been initiated to identify orthopoxvirus virulence genes. The first involves DNA sequence analysis of a 9 kb DNA stretch of vaccinia virus genome previously shown to be non-essential for replication in tissue culture, but important in virus virulence in the mouse. The second involves developing an insertional inactivation mutagenesis system suitable for screening large regions of the ectromelia virus genome for functions important in virus virulence. Using ectromelia virus and two separate experimental approaches, it was shown that recovery from a primary infection did not require the synthesis of anti-ectromelia virus neutralizing antibody. These experiments also showed that a normal anti-ectromelia CTL response could be generated in the absence of L3T4+ ("helper" T-cells). This CTL maturation pathway may be of general importance in resistance to a variety of agents that replicate in cells and express antigens at the cell surface. Genetic analysis of crosses between susceptible (A/J) and resistant (C57BL/6J) mice indicated that at least two non-H2 genes were important in the recovery of mousepox. Studies are aimed at producing a mouse strain A.B6-Rmp (Resistance Mouse Pox).